Vaccinia virus growth in BSC-1 and HeLa cells was inhibited by aphidicolin concentrations of 20 Mu or more. Virus yield and plaque formation in BSC-1 cells were reduced several hundredfold by 80 Mu aphidicolin. The yield was decreased when drug was added early in infection, but was not significantly affected when drug was added at 7.5 h post-infection. Viral inhibition was completely reversed by removing the drug and suspending the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about tenfold by 1 Mu aphidicolin. In cells with replicating vaccinia virus DNA, aphidicolin concentrations over 10 Mu were needed to reduce DNA synthesis to the same extent as in uninfected cells. Aphidicolin inhibition of viral DNA synthesis is removed by washing and suspending infected cells in drug-free medium. Fractionation of infected cells which had been incubated with 1 Mu drug showed that cytoplasmic viral DNA synthesis was relatively resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be mainly from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 60 or 80 Mu aphidicolin did not appear. However, after mutagenesis with hydroxylamine, mutants were generated which did form large plaques in medium with 80 Mu drug. In cells with replicating aphidicoli-resistant virus, DNA synthesis was more than four times more resistant to 80 uM aphidicolin than in cells with replicating wild-type virus. The chromatographic patterns of the viral DNA polymerase isolated from cells with replicating wild-type or resistant virus were similar. However, in an in vitro DNA polymerase assay, 50% inhibition of enzyme activity was obtained with about 75 and 188 Mu aphidicolin for the wild-type and resistant polymerases, respectively. The viral enzymes were much more resistant to the drug than were the cell polymerases. Both intact and Hind-3 digested DNA from the drug-resistant virus were able to rescue aphidicolin-sensitive virus. The resistant DNA has been cloned into a bacterial plasmid.